Epigenetic differences between naïve and primed pluripotent stem cells

Open Access
Review

Abstract

It has been 8 years since the concept of naïve and primed pluripotent stem cell states was first proposed. Both are states of pluripotency, but exhibit slightly different properties. The naïve state represents the cellular state of the preimplantation mouse blastocyst inner cell mass, while the primed state is representative of the post-implantation epiblast cells. These two cell types exhibit clearly distinct developmental potential, as evidenced by the fact that naïve cells are able to contribute to blastocyst chimeras, while primed cells cannot. However, the epigenetic differences that underlie the distinct developmental potential of these cell types remain unclear, which is rather surprising given the large amount of active investigation over the years. Elucidating such epigenetic differences should lead to a better understanding of the fundamental properties of these states of pluripotency and the means by which the naïve-to-primed transition occurs, which may provide insights into the essence of stem cell commitment.

Keywords

Epigenetics Naïve and primed pluripotency Embryonic stem cells (ESCs) Epiblast-derived stem cells (EpiSCs) X-chromosome inactivation (XCI) Three-dimensional (3D) genome organization 

Introduction

In 2007, a new type of stem cells, the epiblast-derived stem cells (EpiSCs), was isolated from the post-implantation epiblast in mice [1, 2]. These pluripotent cells possess features that distinguish them from mouse embryonic stem cells (mESCs). It was an exciting time for the field of stem cell research, as many groups were following up and building on the reprogramming experiments described in the first report of the isolation of induced pluripotent stem cells (iPSCs) via forced expression of four transcription factors in somatic cells [3, 4]. Since the discovery of EpiSCs and techniques for deriving iPSCs occurred within a short time frame, the possibility of multiple stable and metastable pluripotent states soon emerged, eventually leading to the proposal of naïve and primed pluripotent states representing the distinct cellular identities of pre- and post-implantation epiblast cells, respectively [5]. This in turn raised the question of how cells could transition between naïve and primed states, with particular interest in the ‘reverse’ transition from primed-to-naïve state (i.e., reprogramming).

Differences between naïve and primed pluripotency have been extensively studied, from culture conditions and functional capacities to gene expression profiles and chromatin modification states, as outlined in an excellent recent review by Weinberger et al. [6]. However, many of these properties are not readily observable without undergoing days or even weeks of experimental procedures and/or treatments. Examples of such not immediately detectable properties include the long-term dependence of primed (but not naïve) cells on Activin and FGF signaling, and the inability of primed (but not naïve) cells to contribute to blastocyst chimera formation. This raises the question of whether there is a decisive intrinsic difference that demarcates naïve from primed cells that is also readily observable. Epigenetic signatures on their chromatin represent an attractive candidate for study, as these should form the basis for their respective cell-type-specific gene expression programmes and differences in their functional capacities. However, a fuller understanding of these epigenetic differences remains a distant goal. In this review, we discuss the state of the science with regard to pluripotent cell epigenetics and look ahead to potential areas of investigation that might provide new breakthroughs.

We should also note here that there may in fact be a continuum of intermediate states between naïve and primed states in vivo [7]. However, not all such states have been captured in vitro and moreover, even for intermediate states reported to date, their epigenetic status has not been thoroughly analyzed. For this reason, we mainly focus on the naïve and primed states in this article, and subsequently address one intermediate state, the formative pluripotent state, in depth [8].

Brief historical overview of naïve and primed pluripotency

In 1981, it was first reported that mouse embryonic stem cells (mESCs) had been established from the inner cell mass (ICM) of late blastocysts [9, 10]. At the time, teratocarcinoma formation after transplantation into immunodeficient mice was the primary test of cellular pluripotency. In 1984, mESCs were shown to contribute to the formation of chimeric mice after injection into the blastocyst, and this assay then became the gold standard test of pluripotency and remained so for many years [11]. Later, in 1998, human ESCs (hESCs) were isolated from the ICM, and it became clear that the culture conditions used for growing hESCs were distinct from that used for mESCs; the requirement or lack thereof for Activin A and FGF2 being examples of these differences [12, 13, 14]. Moreover, many female hESC lines exhibited an inactive X chromosome (Xi), which suggested that the epigenetic signatures of hESCs were distinct from those of mESCs [15]. Then, in 2007, it was reported that when the post-implantation epiblasts of E5.5 mouse embryos were cultured under the same conditions as those used for hESCs, a new type of stem cell could be isolated; these were named EpiSCs [1, 2]. Indeed, mouse EpiSCs (mEpiSCs) exhibited characteristics similar to hESCs in various aspects such as X-inactivation and poor survival after single-cell suspension. Unlike mESCs, mEpiSCs rarely contributed to chimeric mice [1, 2], which suggested that their developmental potentials are distinct, with mEpiSCs representing a more restricted state.

In a parallel development to this line of work, mouse iPSCs (miPSCs) were established in 2006 [3]. The iPSCs initially reported by Yamanaka and colleagues [3] were considered to be partially reprogrammed because they formed teratomas but failed to contribute to chimeric mice. It did not take long, however, to establish iPSCs capable of contributing to chimera formation [16, 17, 18]. Interestingly, in these more fully reprogrammed ‘standard’ iPSC clones, the Xi was reactivated [18]. In contrast, partially reprogrammed iPSC clones that formed teratomas but failed to contribute to chimeric mice maintained the Xi, suggesting that they had epigenetic signatures distinct from the standard iPSCs [18]. It should be noted that no iPSC is completely reprogrammed epigenetically: for instance, iPSCs retain residual DNA methylation patterns of parental somatic cells, while mESCs generated via somatic cell nuclear transfer exhibit a more complete erasure, resembling that in conventional mESCs [19, 20]. As for mEpiSCs, it was soon discovered that mEpiSCs exhibit an Xi [21]. Thus, in both differentiation and reprogramming, the Xi state appears to be tightly associated with the differentiation state of cells and their developmental potential [22, 23, 24, 25].

Given these findings, it was proposed that there exist two stem cell states with distinct epigenetic signatures, which the authors named naïve and primed pluripotent states [5]. Naïve mESCs are derived from the ICM of the preimplantation blastocyst and are cultured in serum/LIF or 2i/LIF medium (two inhibitors (i) for MEK and GSK3 along with leukemia inhibitory factor LIF), in which they show round, dome-shaped cell colony morphology [6]. Primed mEpiSCs are derived from the post-implantation epiblast and require Activin and FGF signaling [1, 2]. Unlike mESCs, mEpiSC colonies grow as a monolayer and are morphologically similar to hESC colonies [1]. Moreover, mEpiSCs cannot be dissociated down to a single-cell suspension; if this is attempted the cells will undergo apoptosis in a manner dependent on the Rho-associated, coiled-coil containing protein kinase (ROCK) pathway [26]. Both naïve and primed cells utilize the Oct4/Sox2/Nanog transcription factor network, although their genome-wide binding profiles are distinct and their target genes differ slightly between naïve and primed states [6, 27]. Thus, the gene expression profiles of naïve and primed cells are similar, but distinct.

Naïve vs. primed: what could be the most critical epigenetic difference?

Naïve mESCs can be differentiated to a primed mEpiSC-like cell state by culturing mESCs in mEpiSC culture conditions [21, 28]; however, conversion in the opposite direction is challenging, which suggests the presence of some form of epigenetic barrier [21, 29, 30]. One obvious manifestation of developmental potential is the transcriptome, which is similar but distinct in mESCs and mEpiSCs [1, 2]. What could be the epigenetic difference underlying their distinct developmental potentials? In other words, what could be the most critical difference(s) in their chromatin structure?

In a narrow sense, major epigenetic marks as we know them today can be subdivided into two types: histone modifications and DNA methylation. Histone modification patterns are distinct between naïve and primed cells [1]. However, it is difficult to describe all of the differences in histone modifications concisely and pinpoint the ones that are critical. Moreover, it remains controversial whether histone modifications are a cause or a consequence of gene expression patterns. Distinct histone modification patterns on gene promoters may simply reflect their distinct transcription states [31, 32, 33].

Differences in enhancer histone modifications between naïve and primed cells have also been reported [34], and enhancer usage in these cells differs, even for genes expressed in both states [34]. A good example is Oct4 enhancer usage, in which the distal enhancer (DE) is preferentially utilized in the naïve state, whereas the proximal enhancer (PE) is primarily used in the primed state [1, 35]. This distinction implies differences in long-range chromatin interactions, which may contribute to the local three-dimensional (3D) genome organization.

DNA methylation also varies across the naïve and primed states; it has been reported that the genomic DNA of naïve mESCs is generally hypomethylated, whereas in primed mEpiSCs it is hypermethylated [6, 36, 37]. In fact, naïve mESCs cultured in 2i/LIF medium in vitro exhibit a widespread loss of DNA methylation, including genomic imprints [38]. In ‘less naïve’ mESCs grown in serum/LIF medium, the DNA methylation level is clearly higher than that in naïve mESCs grown in 2i/LIF, although not as high as in mEpiSCs [39, 40]. Thus, DNA methylation state is one clear example of epigenetic differences between mESCs and mEpiSCs. Surprisingly however, this difference is observed only in vitro and not in the in vivo counterparts of these cell types; it has been shown that the mouse epiblast cells are globally DNA hypomethylated, both pre- and post-implantation [41]. Thus, any epigenetic difference between mESCs and mEpiSCs may not readily translate to a difference between naïve and primed states in vivo. It is also important to note that a cause–effect relationship has not been established. DNA methylation is enriched in repressed genes in mEpiSCs, but this could merely reflect gene repression [42].

Thus, while various epigenetic marks have been analyzed, it remains to be determined exactly which epigenetic difference demarcates naïve and primed states. Moreover, identifying chromatin modification differences is only the beginning, as the daunting task of addressing whether such differences are a cause or a consequence of their distinct transcription patterns also remains.

Naïve vs. primed: signs of X-chromosome inactivation exist only in the latter

While our initial intent was to widely cover and summarize the current knowledge on the epigenetic differences between naïve and primed states, we came to realize that no crucial epigenetic difference between naïve and primed cells in the form of chromatin modifications has been identified. In the context of embryonic development (the context for which Conrad Waddington first coined the term ‘epigenetics’), mEpiSCs are clearly more advanced than mESCs [43, 44]. Moreover, mESCs contribute to chimeric mice, while mEpiSCs do so only rarely. This strongly suggests differences in their developmental potential, and thus some form of epigenetic signature that is distinct between these two cell types.

Although no clear chromatin differences between naïve and primed cells have been reported, female X-chromosome inactivation (XCI) state could be the epigenetic signature that best indicates the differences in developmental potential between these cell types, and how it changes during differentiation. While XCI is eventually completed in all somatic lineages following the post-implantation epiblast stage, the processes that lead to XCI are regulated in spatiotemporally distinct manners during early embryogenesis [45]. Female mEpiSCs have been shown to exhibit an Xi, as shown by the presence of a H3K27me3 focus [21, 29], whereas mESCs have two active X chromosomes (Xa), and the reprogramming of somatic cells to iPSCs is accompanied by reactivation of the Xi [18]. Forced expression of exogenous Klf4 in mEpiSCs also leads to Xi reactivation [21]. When one differentiates female miPSCs, XCI is initiated and the Xist long noncoding (lnc) RNA and H3K27me3 become enriched on the Xi in differentiating miPSCs [18, 21]. Thus, XCI state is closely linked to the cell’s differentiation state; naïve mESCs/miPSCs lack an Xi and primed mEpiSCs possess one (Fig. 1a).
Fig. 1

Relationship of naïve-to-primed transition and XCI states in mice and humans. a Schematics of the relationship between naïve and primed states and XCI in mice. XaXa represents two active Xs, while XaXi represents the presence of an Xi. In mice, the cells of the ICM of the blastocyst are thought to represent the naïve state in vivo. They exhibit two pinpoint Xist RNAFISH signals (tiny blue dots) inside the nucleus, which indicates that these cells have not initiated XCI. Upon differentiation, the cells likely go through multiple intermediate stages before becoming the late epiblast cells, which have acquired the primed state in vivo and exhibit a single Xist RNA cloud coating the Xi (large blue foci). The naïve state can be captured in vitro in the form of mESCs cultured in medium containing either serum/LIF or 2i/LIF, with the latter showing more uniform naïve properties. Female naïve mESCs exhibit active transcription from both Xs as shown by the uniform yellow fluorescence of female mESCs derived from the Momiji mice [104]. In the Momiji mice, the cells have a CAG promoter-driven eGFP reporter on one X and a mCherry reporter on the other at the same locus, and therefore the cells exhibit yellow fluorescence when the reporters are biallelically expressed, such as in naïve mESCs. The conversion of mESCs to mEpiSCs in vitro may occur via an intermediate stage represented by the ‘formative’ EpiLC state, which has not initiated the XCI and resemble the post-implantation epiblast (E5.75) based on transcriptome data [88]. The primed mEpiSCs derived from the Momiji mice show either green or red fluorescence, indicating that the cells have inactivated one of the two X chromosomes by random XCI. b Schematics of the relationship between naïve and primed states and XCI in humans. The schematic drawing is somewhat speculative, with areas of uncertainty indicated by several question marks. First, there are multiple ‘naïve’ hESCs derived from conventional hESCs by various methods in vitro with slightly different properties including the regulation of XIST lncRNA, which is highly expressed in the 5i/L/A culture condition [78] but not in others [73, 75, 77]. In human blastocysts, cells show biallelic expression of X-linked genes, indicating that they are in an XaXa state, but paradoxically exhibit double XIST RNA cloud accumulation per nuclei [65]. The precise relationship of these various ‘naive’ cells established in vitro and their relationship to the cells of the blastocyst in vivo are still unclear. Upon differentiation, the ICM cells presumably go through a series of intermediate states including those that represent the post-implantation early epiblast (postE-EPI) and late epiblast (postL-EPI), based on a recent study of the early embryogenesis of cynomolgus monkeys [129]

Many regulatory steps lead to the completion of XCI, and XCI states come in different flavors [25, 46] (Fig. 2). For instance, during mESC differentiation in vitro, it is believed that the coating of the future Xi by Xist RNA is one of the earliest events upon initiation of XCI. Afterward, the exclusion of RNA pol II and active histone modifications of the future Xi occur, followed by PRC2 and PRC1 recruitment [47, 48] and the addition of repressive histone marks, H3K27me3 and H3K9me2, to the Xi [49, 50]. Recruitment of macro-H2A and Ash2L are considered to be rather late events in XCI [51, 52], as is the chromosome-wide replication timing switch from the early to late S-phase of the Xi [53, 54]. The XCI mark used to define XCI in a given report thus warrants close attention. For instance, if H3K27me3 foci on the Xi are used as a mark to define XCI and were detected in a cell type analyzed, this does not guarantee that the Xi in this cell type had completed the XCI events downstream of H3K27me3 foci formation. It should also be noted that the order of events described above are based on analyses of cell populations during mESC differentiation [46], but there is no strong evidence of whether this order holds true in individual cells or during mouse embryogenesis.
Fig. 2

A rough outline of the temporal relationship of various epigenetic marks associated with the Xi based on mESC differentiation studies [46]. There are so many Xi-associated marks that have been and are being discovered that it is not entirely clear which set is conserved across species. It is also not clear which marks are present in primed and formative pluripotent cells

Interestingly, we recently witnessed the discovery of a wide variety of Xist RNA-binding proteins, which together built a foundation for dissecting the earliest phase of heterochromatin formation on the Xi [55, 56, 57, 58, 59]. Meanwhile, the rise of Hi-C (a genome-wide, high-throughput chromosome conformation capture method) [60] and its application to XCI studies has led to the discovery that the Xi is subdivided into two ‘megadomains,’ which are several tens of megabases in size [57, 61, 62], as well as that TAD (topologically associating domain) boundaries disappear from the Xi upon XCI [63]. Thus, there are already many novel Xi-specific marks that are waiting to be characterized, and more will certainly be identified in the future. Where each of these marks lies in the context of the multi-step XCI process, their causal relationship, and how these marks relate to the primed state are important areas of future investigation in the effort to better understand the epigenetic state of the Xi in primed cells (Fig. 2).

Conserved features of the X-chromosome inactivation process and the 3D genome organization

XCI regulation in mice is fairly complex, but in other species it is even more complicated [64]. Analyses of XCI in humans and rabbits have revealed that the XCI processes are markedly different between rodents and other eutherians. For example, unlike in mice, human Xist RNA is expressed from and coats both X chromosomes in females in the ICM of the early blastocyst, and this is maintained even in late blastocysts (Fig. 1b) [65]. Similarly, ~ 25% of early blastocyst cells in female rabbits show two Xist RNA clouds, although one of the two clouds disappears by the late blastocyst stage. Thus, the initial Xist coating of X chromosomes in human and rabbit ICM cells is unrelated to their future XCI fate. Meanwhile, human and rabbit female somatic cells exhibit a single Xi with an Xist cloud, meaning that the two Xist clouds on both X chromosomes eventually become localized only to the Xi at some point during development. How cells achieve this is currently unknown. Because lagomorphs (rabbits) are more closely related to rodents (mice) than any other mammals and yet show more similarity to primates (humans) with respect to XCI regulation, it could be argued that rodents are the exception rather than the rule [65]. These observations highlight the importance of understanding XCI regulatory processes in non-rodent organisms, and of distinguishing the processes that are conserved across species from those that are specific to certain organisms. Such comparative analysis would help us to identify the conserved core regulatory mechanisms of XCI and to elucidate conserved Xi-specific epigenetic signatures that demarcate primed from naïve states.

Interestingly, one of the most conserved properties of the Xi is its replication late in S-phase [54, 66, 67], which is observed not only in eutherians but also in marsupials and monotremes [68]. In female mESCs, the two X chromosomes show early replication, while in female mEpiSCs the Xi replicates late [69], consistent with the Xi becoming late replicating after the post-implantation epiblast stage in vivo [53]. DNA replication timing has recently been shown to be closely associated with the 3D genome organization [69, 70, 71], and whether or not the early to late replication timing shift of the Xi reflects a 3D organizational change is of interest. As discussed earlier, allele-specific Hi-C analysis has led to the elucidation of the 3D organization of the Xi, which exhibits two ‘megadomains’ and lacks TAD boundaries in somatic cells, such as fibroblasts, brain cells, and neural progenitor cells [57, 61, 62, 63, 72]. It remains unknown whether these novel properties of the Xi are observed in primed cells, and how they relate to the compact Xi structure, or the Barr body. Moreover, naïve and primed cells exhibit distinct enhancer usage for some genes, which suggests differences in long-range chromatin interaction [34]. Differences in the 3D genome organization between naïve and primed cells are an interesting yet relatively unexplored area and may provide us with clues into the broader implications of the Xi structure observed only in females.

Naïve pluripotent state in human cells?

Conventional human ESCs often exhibit signs of XCI and high Oct4 PE enhancer activity, and because their culture condition is similar to mouse EpiSCs they have been regarded as primed cells [6]. Whether such a state as naïve hESCs exists is unclear [6], but attempts have been made to establish naïve hESCs to directly address whether or not the naïve state exists in human cells. By 2014, several groups had reported the establishment of ‘naïve’ hESCs [73, 74, 75, 76, 77, 78, 79, 80]; these show gene expression profiles more closely resembling those of mESCs than the conventional hESCs, based on a clustering analysis of their transcriptomes [73, 74, 75, 76, 77, 78, 79, 80]. Certain differences have been found between these ‘naïve’ hESCs and mESCs, however. For instance, two distinct cis-regulatory elements of Oct4, DE and PE, are active primarily in naïve and primed states, respectively, in mice and these various human ‘naïve’ cells do exhibit high DE activity. However, DE activity was often only transient and most ‘naïve’ cells eventually switched to activate PE upon long-term culture. The only exception was the 5i/L/A culture condition reported by Theunissen et al. [78]. However, 5i/L/A culture caused an erasure of DNA methylation in regions subject to genomic imprinting [81], which would be a problem in regenerative medicine. Furthermore, XCI states were variable among these ‘naïve’ cells, with some showing two XIST clouds on both X chromosomes, but no clouds in others (Fig. 1b). DNA methylation states of the XIST promoter also vary among ‘naïve’ cells, with some showing higher methylation levels than others. These issues have to be resolved in parallel with the elucidation of the conserved features of the XCI processes. In any case, the pursuit of a ‘gold standard’ method for maintaining naïve hESCs in culture is certain to continue [82].

Regarding hESCs, a peculiar phenomenon called erosion of XCI has been observed [83, 84]. On long-term culture of female hESCs, the XCI marks on the Xi, namely XIST cloud and H3K27me3, disappear and transcriptional activity is derepressed on the Xi [83]. When this XCI erosion occurs, XACT lncRNA, which normally coats the Xa, covers the Xi prior to it losing its XIST RNA coating [85, 86]. This is particularly troublesome, as once hESCs/hiPSCs experience XCI erosion, they never undergo XCI again, even if they are differentiated [86]. This is clearly distinct from the Xi reactivation phenomenon that occurs during reprogramming, and poses a safety issue when hESCs/hiPSCs are used as source cells in the development of regenerative medicine. We clearly need to accumulate more knowledge on culture conditions that can stably maintain epigenetic states of naïve or primed hESCs/hiPSCs in a controlled manner. For instance, a very recent study revealed that hiPSCs with an eroded Xi can still undergo XCI upon differentiation if the cells are converted to a ‘naïve’ state [78] prior to differentiation [87], implying that there are ways to reset the eroded state. These authors also revealed that ‘naïve’ hESCs derived from the blastocyst and from conventional (i.e., primed) hESCs (by 5iLAF culture similar to 5i/L/A [78] but with FGF2) both exhibited two active Xs and yet only the former exhibited one or two XIST RNA clouds [87]. Interestingly, ‘naïve’ hESCs derived from conventional hESCs formed an XIST RNA cloud on a single X after adaptation in 5iLAF for several passages, suggesting that they became more similar to ‘naïve’ hESCs directly derived from the blastocyst [87].

New tools to approach and monitor naïve and primed pluripotency and their transitions

In human cells, it is difficult to describe the epigenetic differences between naïve and primed cells/states simply because, as discussed earlier, the human naïve state is not fully understood. Many different ‘naïve’ human cells have been proposed, and at present it is impossible to say which one corresponds to the naïve state in mice (Fig. 1b). Addressing this is important not only for human stem cell biology, but also for the understanding of evolutionarily conserved aspects of the epigenetic differences between naïve and primed cells. Another important challenge is to understand the processes by which naïve cells acquire the primed pluripotent state during differentiation. However, several research advances and new technologies have been reported in this area.

First, a distinct type of stem cells named EpiLCs (epiblast-like cells) was successfully derived from mESC differentiation in vitro [88]. Although it has not been possible to stably maintain EpiLCs, they can be generated reproducibly and relatively easily by differentiating naïve mESCs for 2 days in adherent culture using defined medium conditions [8, 89]. Mouse EpiLCs exhibit gene expression patterns that closely resemble the early epiblast in vivo and serve as an excellent substrate to generate primordial germ cell-like cells (PGCLC) in vitro, while mESCs and mEpiSCs do not [88, 90]. Moreover, female mouse EpiLCs lack H3K27me3 enrichment on the future Xi, which indicates that XCI either has not initiated or at least is far from completion in these cells [90] (Fig. 1a). Thus, mouse EpiLCs represent a unique differentiation stage somewhere in between the naïve and primed states; their gene expression profile has clearly shifted from a mESC-like to an epiblast-like state and resemble the early primitive ectoderm-like (EPL) cells [91] or Rex1-negative mESCs [92], but their epigenetic state is still closer to the mESCs. The EpiLC state was recently designated the formative pluripotency state [93], and EpiLCs provide the unique opportunity to scrutinize known properties of naïve and primed states and see when they change during naïve–formative–primed transitions or whether they are exclusively associated with the naïve or primed states [89, 94, 95, 96, 97].

Rex1/Zfp42 is one of the representative ICM genes that are sharply downregulated upon exit from naïve state during mESC differentiation. Austin Smith’s group generated a mESC line in which a transgene encoding GFP with a half-life of 2 h was knocked into the Rex1 locus [98]. When cultured in the 2i condition, mESCs were Rex1-positive, whereas in serum/LIF medium without 2i, GFP (Rex1)-positive and negative cell populations coexisted, allowing them to focus on the earliest phase of differentiation in which the mESCs exit the naïve state [99]. This system, combined with haploid mESCs [100, 101, 102], which are an excellent tool for forward genetics, was used to screen for factors required for the cells to exit the naïve state [99].

Recently, Choi et al. generated mESCs derived from Oct4-ΔPE-GFP and Oct4-ΔDE-RFP double transgenic mice [103]. In mouse embryos, the cells were GFP positive (i.e., DE positive) until the blastocyst stage and became GFP/RFP double positive after E5.5 (i.e., DE and PE positive), whereas in mESCs cultured in vitro, the cells were GFP positive in 2i/LIF condition and GFP/RFP double positive in serum/LIF. Interestingly, Choi et al.’s mEpiLCs derived from these mESCs became RFP positive [103]. However, their EpiLCs, designated as EpiSC-like cells, went through multiple passages and were clearly not the equivalent of the EpiLCs described by Hayashi et al. [88]. It will be interesting to see whether the EpiLCs reported by Hayashi et al. utilize Oct4-DE, -PE, or both regulatory elements.

Kobayashi et al. recently established a novel X-linked eGFP/mCherry dual reporter mouse strain, named Momiji (named after the autumn leaves of Japanese maple trees), which enables real-time monitoring of the XCI state during mouse development [104]. The first mice with an X-linked GFP reporter, X-GFP, were established by Hadjantonakis et al. [105]. These X-GFP mice were used to establish GFP-negative EpiSCs, which, when cultured in LIF+ medium for a few weeks, generated GFP-positive cells, providing a rare example of spontaneous primed-to-naïve conversion [29]. In Momiji mice, CAG promoter-driven eGFP and mCherry are knocked into the maternal and paternal Hprt locus, respectively (or vice versa in reciprocal mice) [104], which allows simultaneous monitoring of both X chromosomes. The same system was built into the Pgk1 locus as well. In either case, green or red indicates random XCI, while yellow indicates the presence of two active Xs in ESCs or upon Xi reactivation (Fig. 1a). How well Hprt and Pgk1 loci represent the chromosome-wide transcriptional activity of the entire X chromosomes is a matter of debate, but with the Momiji mice the XCI state can now be monitored live in early mouse embryogenesis and during mESC differentiation in vitro [104]. How the changes in the XCI state relate to the cell fate transitions during mouse embryogenesis is a challenge for the future that could be addressed with this system.

Future directions: new approaches, single-cell epigenomics, and live-cell imaging

A growing body of evidence suggests that there must be a set of epigenetic marks, both known and unknown, that contributes to the crucial difference between naïve and primed pluripotent cells, but we are still only halfway through the journey to understanding. We still need to precisely describe various epigenetic events and clarify their causal and temporal relationships one by one. In doing so, at least three important issues come to mind.

First, we were interested to note that many of the studies reviewed in preparing this article relied on a limited number of markers when distinguishing between naïve and primed states. In general, there may be an over-reliance on the differential usage of Oct4-DE and -PE, which is merely a single gene regulatory event. Moreover, the response of these enhancers is clearly not all-or-none, indicating the importance for future studies of cautiously determining the pluripotency state by examining additional features. This trend may be a reflection of how little is known about the epigenetic differences between naïve and primed states. The field should continue to search for additional reliable markers that can clearly distinguish the two states.

In this regard, one emerging area of interest is the role of energy metabolism in regulating the epigenetic status of naïve and primed cells [106, 107]. In a seminal study, Zhou et al. reported that naïve mESCs rely on both anaerobic (glycolytic) and aerobic (mitochondrial) respiration, while primed mEpiSCs rely almost exclusively on glycolysis [108]. Importantly, this metabolic difference is observed in vivo in the context of the transition from the ICM of the mouse blastocyst to the post-implantation epiblast [108], as well as conserved in the context of naïve vs. primed human ESCs/iPSCs [77, 109]. These observations provoked interest in the potential roles of various metabolites in regulating the epigenetic states in naïve and primed cells and led to, for instance, the discovery of the role of α-ketoglutarate, a TCA (tricarboxylic acid) cycle intermediate, in maintaining naïve pluripotency through promoting histone/DNA demethylation [110], while accelerating differentiation of primed mouse EpiSCs and human ESCs [106]. Nicotinamide N-methyltransferase (NNMT), which controls the amount of S-adenosyl methionine (SAM) available for H3K27me3, is required to maintain low H3K27me3 levels and keeps the Wnt pathway active and the HIF pathway inactive, helping hESCs to sustain their ‘naïve’ state [109]. Maintenance of a constant SAM level in contrast is crucial to the self-renewal of human ESCs/iPSCs [111].

Secondly, we will need a good in vitro ESC differentiation system that is homogeneous and synchronous, which should help in elucidating the order of various events that eventually lead to the formation of primed cells originating from naïve cells, and enables next-generation sequencing (NGS)-based epigenomic analyses, even with cell populations. In recent years, many excellent ESC/iPSC differentiation protocols have been developed to reconstitute certain lineage differentiations in vitro and generate tissue organoids. Many of these protocols rely on insights obtained from basic developmental biology over the years to recapitulate germ layer and tissue differentiation in a stepwise manner in vitro [90, 112, 113]. The earliest steps of these sophisticated protocols may yield clues on improving early differentiation processes relevant to the naïve-primed transition, which was exactly the case with EpiLCs and PGC development [88].

In addition, continuous efforts to improve ESC culture conditions may also be important. For example, Nichols and Smith first proposed that the epiblast in vivo constitutes the ‘ground state,’ meaning a fully unrestricted population that harbors the requisite developmental potency and flexibility to produce all embryonic lineages [5], and the term ‘ground state’ has also been used to describe the developmental state of naïve mESCs cultured in 2i/LIF medium in vitro [7]. However, Yagi et al. recently reported that DNA methylation imprints are erased in female mESCs grown in 2i/LIF and these cells also exhibited impaired autonomous embryonic and placental development as assayed by tetraploid embryo complementation or somatic cell nuclear transfer [38]. This warrants reconsideration of the definition of ‘ground state’ pluripotency in vitro and underscores the potential of the ‘alternative 2i (a2i)’ approach with the Mek1/2 inhibitor replaced by a Src inhibitor CGP77675, which can preserve the epigenetic stability of genomic imprints and the developmental potential of early passage female mESCs [38]. It should be noted that the a2i approach is not perfect and problems can arise upon prolonged culture of female mESCs, but this approach should certainly stimulate the field.

The third key issue is in vivo analysis. Once differential properties between naïve and primed cells are identified, it is essential to address whether those differences are also observable in vivo. DNA methylation is a classic example in which behaviors in the dish differ from those in the body [41]. Moreover, as in the case of XCI, the more embryonic tissues and species analyzed, the more unambiguous the distinction will be between conserved and species-specific properties of naïve and primed states. In general, however, in vivo analysis is challenging. Conventional cell-based assays that utilize fluorescent labeling such as immunostaining and FISH-based approaches are feasible, but to perform time-course analyses some form of a live imaging system is preferred. These methods do not allow visualization of genome-scale properties, while single-cell NGS approaches do. Single-cell epigenome profiling by NGS, however, is still challenging. Many single-cell epigenome profiling methods have been reported, e.g., for histone modifications [114], DNA methylation [115, 116], ATAC-seq [117], Hi-C [118, 119, 120], Dam-ID [121], and so on [122], but only a few have been applied to the analysis of embryonic cells in vivo due to various issues, including cost, resolution, and technical difficulties [120, 123, 124, 125]. Serious efforts are now being made, but there are still few single-cell, NGS-based epigenome profiling methods that are sufficiently reliable for use in the analysis of embryonic cells in vivo.

However, the situation is different for RNA-seq analyses. Various single-cell RNA-seq protocols have been established that are practical and reliable enough to be applied to the analysis of embryonic cells in vivo. Furthermore, the analytical platform has gradually shifted from multi-well plates to microfluidics to droplet-based technologies, which will lead to new and important discoveries regarding the behaviors and properties of single cells within large cell populations [126, 127, 128]. The timing at which chromosome-wide silencing of the Xi takes place in mice, or the switch from imprinted to random XCI during the morula–blastocyst–epiblast transition, may be amenable to single-cell RNA-seq analysis. A recent single-cell RNA-seq study of early embryogenesis in the cynomolgus monkeys has demonstrated the power of in vivo analysis and built a foundation for discovering conserved features of the naïve-to-primed transition in vivo [129] (Fig. 1b). Furthermore, the use of cynomolgus monkeys allows us to test the ability of cells to contribute to chimeric animals, which should help in revealing the conserved features of the naïve and primed states [130].

These novel approaches and their future developments, combined with steady efforts to elucidate the causal and temporal relationships between different properties of naïve and primed cells, should gradually reveal their critical intrinsic differences. Moreover, such efforts may lead to the identification or in vitro capture of additional intermediate states in between naïve and primed, perhaps akin to the manner in which the EpiLCs were identified. Through these efforts, the next frontier in this field should emerge.

Notes

Acknowledgements

We thank S. Keeley, D. Sipp, and anonymous reviewers for helpful comments on the manuscript. This work was supported by a RIKEN CDB Intramural Grant to I.H. and Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology (17H05597, 17K07498) to S.K.

References

  1. 1.
    Tesar PJ, Chenoweth JG, Brook FA, Davies TJ, Evans EP, Mack DL, Gardner RL, McKay RD (2007) New cell lines from mouse epiblast share defining features with human embryonic stem cells. Nature 448:196–199PubMedCrossRefGoogle Scholar
  2. 2.
    Brons IGM, Smithers LE, Trotter MWB, Rugg-Gunn P, Sun B, de Sousa Chuva, Lopes SM, Howlett SK, Clarkson A, Ahrlund-Richter L, Pedersen RA, Vallier L (2007) Derivation of pluripotent epiblast stem cells from mammalian embryos. Nature 448:191–195PubMedCrossRefGoogle Scholar
  3. 3.
    Takahashi K, Yamanaka S (2006) Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126:663–676PubMedCrossRefGoogle Scholar
  4. 4.
    Takahashi K, Yamanaka S (2016) A decade of transcription factor-mediated reprogramming to pluripotency. Nat Rev Mol Cell Biol 17:183–193PubMedCrossRefGoogle Scholar
  5. 5.
    Nichols J, Smith A (2009) Naive and primed pluripotent states. Cell Stem Cell 4:487–492PubMedCrossRefGoogle Scholar
  6. 6.
    Weinberger L, Ayyash M, Novershtern N, Hanna JH (2016) Dynamic stem cell states: naive to primed pluripotency in rodents and humans. Nat Rev Mol Cell Biol 17:155–169PubMedCrossRefGoogle Scholar
  7. 7.
    Morgani S, Nichols J, Hadjantonakis A-K (2017) The many faces of pluripotency: in vitro adaptations of a continuum of in vivo states. BMC Dev Biol 17:7PubMedPubMedCentralCrossRefGoogle Scholar
  8. 8.
    Hayashi K, Saitou M (2013) Generation of eggs from mouse embryonic stem cells and induced pluripotent stem cells. Nat Protoc 8:1513–1524PubMedCrossRefGoogle Scholar
  9. 9.
    Evans MJ, Kaufman MH (1981) Establishment in culture of pluripotential cells from mouse embryos. Nature 292:154–156PubMedCrossRefGoogle Scholar
  10. 10.
    Martin GR (1981) Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells. Proc Natl Acad Sci USA 78:7634–7638PubMedPubMedCentralCrossRefGoogle Scholar
  11. 11.
    Bradley A, Evans M, Kaufman MH, Robertson E (1984) Formation of germ-line chimaeras from embryo-derived teratocarcinoma cell lines. Nature 309:255–256PubMedCrossRefGoogle Scholar
  12. 12.
    Thomson JA, Itskovitz-eldor J, Shapiro SS, Waknitz MA, Swiergiel JJ, Marshall VS, Jones JM (1998) Embryonic stem cell lines derived from human blastocysts. Science 282:1145–1148PubMedCrossRefGoogle Scholar
  13. 13.
    Vallier L, Alexander M, Pedersen RA (2005) Activin/Nodal and FGF pathways cooperate to maintain pluripotency of human embryonic stem cells. J Cell Sci 118:4495–4509PubMedCrossRefGoogle Scholar
  14. 14.
    Beattie GM, Lopez AD, Bucay N, Hinton A, Firpo MT, King CC, Hayek A (2005) Activin A maintains pluripotency of human embryonic stem cells in the absence of feeder layers. Stem Cells 23:489–495PubMedCrossRefGoogle Scholar
  15. 15.
    Hoffman LM, Hall L, Batten JL, Young H, Pardasani D, Baetge EE, Lawrence J, Carpenter MK (2005) X-inactivation status varies in human embryonic stem cell lines. Stem Cells 23:1468–1478PubMedCrossRefGoogle Scholar
  16. 16.
    Okita K, Ichisaka T, Yamanaka S (2007) Generation of germline-competent induced pluripotent stem cells. Nature 448:313–318PubMedCrossRefGoogle Scholar
  17. 17.
    Wernig M, Meissner A, Foreman R, Brambrink T, Ku M, Hochedlinger K, Bernstein BE, Jaenisch R (2007) In vitro reprogramming of fibroblasts into a pluripotent ES-cell-like state. Nature 448:318–324PubMedCrossRefGoogle Scholar
  18. 18.
    Maherali N, Sridharan R, Xie W, Utikal J, Eminli S, Arnold K, Stadtfeld M, Yachechko R, Tchieu J, Jaenisch R, Plath K, Hochedlinger K (2007) Directly reprogrammed fibroblasts show global epigenetic remodeling and widespread tissue contribution. Cell Stem Cell 1:55–70PubMedCrossRefGoogle Scholar
  19. 19.
    Kim K, Doi A, Wen B, Ng K, Zhao R, Cahan P, Kim J, Aryee MJ, Ji H, Ehrlich LIR, Yabuuchi A, Takeuchi A, Cunniff KC, Hongguang H, Mckinney-Freeman S, Naveiras O, Yoon TJ, Irizarry RA, Jung N, Seita J, Hanna J, Murakami P, Jaenisch R, Weissleder R, Orkin SH, Weissman IL, Feinberg AP, Daley GQ (2010) Epigenetic memory in induced pluripotent stem cells. Nature 467:285–290PubMedPubMedCentralCrossRefGoogle Scholar
  20. 20.
    Ma H, Morey R, O’Neil RC, He Y, Daughtry B, Schultz MD, Hariharan M, Nery JR, Castanon R, Sabatini K, Thiagarajan RD, Tachibana M, Kang E, Tippner-Hedges R, Ahmed R, Gutierrez NM, Van Dyken C, Polat A, Sugawara A, Sparman M, Gokhale S, Amato P, Wolf D, Ecker JR, Laurent LC, Mitalipov S (2014) Abnormalities in human pluripotent cells due to reprogramming mechanisms. Nature 511:177–183PubMedPubMedCentralCrossRefGoogle Scholar
  21. 21.
    Guo G, Yang J, Nichols J, Hall JS, Eyres I, Mansfield W, Smith A (2009) Klf4 reverts developmentally programmed restriction of ground state pluripotency. Development 136:1063–1069PubMedPubMedCentralCrossRefGoogle Scholar
  22. 22.
    Payer B, Lee JT, Namekawa SH (2011) X-inactivation and X-reactivation: epigenetic hallmarks of mammalian reproduction and pluripotent stem cells. Hum Genet 130:265–280PubMedPubMedCentralCrossRefGoogle Scholar
  23. 23.
    Papp B, Plath K (2011) Reprogramming to pluripotency: stepwise resetting of the epigenetic landscape. Cell Res 21:486–501PubMedPubMedCentralCrossRefGoogle Scholar
  24. 24.
    Ohhata T, Wutz A (2013) Reactivation of the inactive X chromosome in development and reprogramming. Cell Mol Life Sci 70:2443–2461PubMedCrossRefGoogle Scholar
  25. 25.
    Chaligné R, Heard E (2014) X-chromosome inactivation in development and cancer. FEBS Lett 588:2514–2522PubMedCrossRefGoogle Scholar
  26. 26.
    Watanabe K, Ueno M, Kamiya D, Nishiyama A, Matsumura M, Wataya T, Takahashi JB, Nishikawa S, Nishikawa S, Muguruma K, Sasai Y (2007) A ROCK inhibitor permits survival of dissociated human embryonic stem cells. Nat Biotechnol 25:681–686PubMedCrossRefGoogle Scholar
  27. 27.
    Galonska C, Ziller MJ, Karnik R, Meissner A (2015) Ground state conditions induce rapid reorganization of core pluripotency factor binding before global epigenetic reprogramming. Cell Stem Cell 17:462–470PubMedPubMedCentralCrossRefGoogle Scholar
  28. 28.
    Greber B, Wu G, Bernemann C, Joo JY, Han DW, Ko K, Tapia N, Sabour D, Sterneckert J, Tesar P, Schöler HR (2010) Conserved and divergent roles of FGF signaling in mouse epiblast stem cells and human embryonic stem cells. Cell Stem Cell 6:215–226PubMedCrossRefGoogle Scholar
  29. 29.
    Bao S, Tang F, Li X, Hayashi K, Gillich A, Lao K, Surani MA (2009) Epigenetic reversion of post-implantation epiblast to pluripotent embryonic stem cells. Nature 461:1292–1295PubMedCrossRefGoogle Scholar
  30. 30.
    Silva J, Nichols J, Theunissen TW, Guo G, van Oosten AL, Barrandon O, Wray J, Yamanaka S, Chambers I, Smith A (2009) Nanog is the gateway to the pluripotent ground state. Cell 138:722–737PubMedPubMedCentralCrossRefGoogle Scholar
  31. 31.
    Ptashne M (2013) Epigenetics: core misconcept. Proc Natl Acad Sci USA 110:7101–7103PubMedPubMedCentralCrossRefGoogle Scholar
  32. 32.
    Henikoff S, Shilatifard A (2011) Histone modification: cause or cog? Trends Genet 27:389–396PubMedCrossRefGoogle Scholar
  33. 33.
    Rando OJ (2012) Combinatorial complexity in chromatin structure and function: revisiting the histone code. Curr Opin Genet Dev 22:148–155PubMedPubMedCentralCrossRefGoogle Scholar
  34. 34.
    Factor DC, Corradin O, Zentner GE, Saiakhova A, Song L, Chenoweth JG, McKay RD, Crawford GE, Scacheri PC, Tesar PJ (2014) Epigenomic comparison reveals activation of “seed” enhancers during transition from naive to primed pluripotency. Cell Stem Cell 14:854–863PubMedPubMedCentralCrossRefGoogle Scholar
  35. 35.
    Yeom YI, Fuhrmann G, Ovitt CE, Brehm A, Ohbo K, Gross M, Hübner K, Schöler HR, Hubner K, Scholer HR (1996) Germline regulatory element of Oct-4 specific for the totipotent cycle of embryonal cells. Development 122:881–894PubMedGoogle Scholar
  36. 36.
    Hackett JA, Dietmann S, Murakami K, Down TA, Leitch HG, Surani MA (2013) Synergistic mechanisms of DNA demethylation during transition to ground-state pluripotency. Stem Cell Rep 1:518–531CrossRefGoogle Scholar
  37. 37.
    Hayashi K, de Lopes SMC, Tang F, Surani MA (2008) Dynamic equilibrium and heterogeneity of mouse pluripotent stem cells with distinct functional and epigenetic states. Cell Stem Cell 3:391–401PubMedCrossRefGoogle Scholar
  38. 38.
    Yagi M, Kishigami S, Tanaka A, Semi K, Mizutani E, Wakayama S, Wakayama T, Yamamoto T, Yamada Y (2017) Derivation of ground-state female ES cells maintaining gamete-derived DNA methylation. Nature 548:224–227PubMedGoogle Scholar
  39. 39.
    Habibi E, Brinkman AB, Arand J, Kroeze LI, Kerstens HHD, Matarese F, Lepikhov K, Gut M, Brun-Heath I, Hubner NC, Benedetti R, Altucci L, Jansen JH, Walter J, Gut IG, Marks H, Stunnenberg HG (2013) Whole-genome bisulfite sequencing of two distinct interconvertible DNA methylomes of mouse embryonic stem cells. Cell Stem Cell 13:360–369PubMedCrossRefGoogle Scholar
  40. 40.
    von Meyenn F, Iurlaro M, Habibi E, Liu NQ, Salehzadeh-Yazdi A, Santos F, Petrini E, Milagre I, Yu M, Xie Z, Kroeze LI, Nesterova TB, Jansen JH, Xie H, He C, Reik W, Stunnenberg HG (2016) Impairment of DNA methylation maintenance is the main cause of global demethylation in naive embryonic stem cells. Mol Cell 62:848–861CrossRefGoogle Scholar
  41. 41.
    Veillard A-C, Marks H, Bernardo AS, Jouneau L, Laloë D, Boulanger L, Kaan A, Brochard V, Tosolini M, Pedersen R, Stunnenberg H, Jouneau A (2014) Stable methylation at promoters distinguishes epiblast stem cells from embryonic stem cells and the in vivo epiblasts. Stem Cells Dev 23:2014–2029PubMedPubMedCentralCrossRefGoogle Scholar
  42. 42.
    Schübeler D (2015) Function and information content of DNA methylation. Nature 517:321–326PubMedCrossRefGoogle Scholar
  43. 43.
    Waddington CH (1957) The strategy of the genes. George Allen Unwin, LondonGoogle Scholar
  44. 44.
    Takahashi K, Yamanaka S (2015) A developmental framework for induced pluripotency. Development 142:3274–3285PubMedCrossRefGoogle Scholar
  45. 45.
    Augui S, Nora EP, Heard E (2011) Regulation of X-chromosome inactivation by the X-inactivation centre. Nat Rev Genet 12:429–442PubMedCrossRefGoogle Scholar
  46. 46.
    Morey C, Avner P (2011) The demoiselle of X-inactivation: 50 years old and as trendy and mesmerising as ever. PLoS Genet 7:e1002212PubMedPubMedCentralCrossRefGoogle Scholar
  47. 47.
    Silva J, Mak W, Zvetkova I, Appanah R, Nesterova TB, Webster Z, Peters AHFM, Jenuwein T, Otte AP, Brockdorff N (2003) Establishment of histone H3 methylation on the inactive X chromosome requires transient recruitment of Eed-Enx1 polycomb group complexes. Dev Cell 4:481–495PubMedCrossRefGoogle Scholar
  48. 48.
    Plath K, Fang J, Mlynarczyk-Evans SK, Cao R, Worringer KA, Wang H, dela Cruz C, Otte A, Panning B, Zhang Y (2003) Role of histone H3 lysine 27 methylation in X inactivation. Science 300:131–135PubMedCrossRefGoogle Scholar
  49. 49.
    Heard E, Rougeulle C, Arnaud D, Avner P, Allis CD, Spector DL (2001) Methylation of histone H3 at Lys-9 Is an early mark on the X chromosome during X inactivation. Cell 107:727–738PubMedCrossRefGoogle Scholar
  50. 50.
    Peters AHFM, Mermoud JE, O’Carroll D, Pagani M, Schweizer D, Brockdorff N, Jenuwein T (2002) Histone H3 lysine 9 methylation is an epigenetic imprint of facultative heterochromatin. Nat Genet 30:77–80PubMedCrossRefGoogle Scholar
  51. 51.
    Costanzi C, Stein P, Worrad DM, Schultz RM, Pehrson JR (2000) Histone macroH2A1 is concentrated in the inactive X chromosome of female preimplantation mouse embryos. Development 127:2283–2289PubMedGoogle Scholar
  52. 52.
    Pullirsch D, Härtel R, Kishimoto H, Leeb M, Steiner G, Wutz A (2010) The Trithorax group protein Ash2l and Saf-A are recruited to the inactive X chromosome at the onset of stable X inactivation. Development 137:935–943PubMedPubMedCentralCrossRefGoogle Scholar
  53. 53.
    Takagi N, Sugawara O, Sasaki M (1982) Regional and temporal changes in the pattern of X-chromosome replication during the early post-implantation development of the female mouse. Chromosoma 85:275–286PubMedCrossRefGoogle Scholar
  54. 54.
    Koren A, Mccarroll SA (2014) Random replication of the inactive X chromosome. Genome Res 24:64–69PubMedPubMedCentralCrossRefGoogle Scholar
  55. 55.
    McHugh CA, Chen C-K, Chow A, Surka CF, Tran C, McDonel P, Pandya-Jones A, Blanco M, Burghard C, Moradian A, Sweredoski MJ, Shishkin AA, Su J, Lander ES, Hess S, Plath K, Guttman M (2015) The Xist lncRNA interacts directly with SHARP to silence transcription through HDAC3. Nature 521:232–236PubMedPubMedCentralCrossRefGoogle Scholar
  56. 56.
    Chu C, Zhang QC, Da Rocha ST, Flynn RA, Bharadwaj M, Calabrese JM, Magnuson T, Heard E, Chang HY (2015) Systematic discovery of Xist RNA binding proteins. Cell 161:404–416PubMedPubMedCentralCrossRefGoogle Scholar
  57. 57.
    Minajigi A, Froberg J, Wei C, Sunwoo H, Kesner B, Colognori D, Lessing D, Payer B, Boukhali M, Haas W, Lee JT (2015) A comprehensive Xist interactome reveals cohesin repulsion and an RNA-directed chromosome conformation. Science 349:aab2276CrossRefGoogle Scholar
  58. 58.
    Moindrot B, Cerase A, Coker H, Masui O, Grijzenhout A, Pintacuda G, Schermelleh L, Nesterova TB, Brockdorff N (2015) A pooled shRNA screen identifies Rbm15, Spen, and Wtap as factors required for Xist RNA-mediated silencing. Cell Rep 12:562–572PubMedPubMedCentralCrossRefGoogle Scholar
  59. 59.
    Monfort A, Di Minin G, Postlmayr A, Freimann R, Arieti F, Thore S, Wutz A (2015) Identification of Spen as a crucial factor for Xist function through forward genetic screening in haploid embryonic stem cells. Cell Rep 12:554–561PubMedPubMedCentralCrossRefGoogle Scholar
  60. 60.
    Lieberman-aiden E, Van Berkum NL, Williams L, Imakaev M, Ragoczy T, Telling A, Amit I, Lajoie BR, Sabo PJ, Dorschner MO, Sandstrom R, Bernstein B, Bender MA, Groudine M, Gnirke A, Stamatoyannopoulos J, Mirny LA, Lander ES, Dekker J (2009) Comprehensive mapping of long-range interactions reveals folding principles of the human genome. Science 326:289–293PubMedPubMedCentralCrossRefGoogle Scholar
  61. 61.
    Deng X, Ma W, Ramani V, Hill A, Yang F, Ay F, Berletch JB, Blau CA, Shendure J, Duan Z, Noble WS, Disteche CM (2015) Bipartite structure of the inactive mouse X chromosome. Genome Biol 16:1–21CrossRefGoogle Scholar
  62. 62.
    Darrow EM, Huntley MH, Dudchenko O, Stamenova EK, Durand NC, Sun Z, Huang S-C, Sanborn AL, Machol I, Shamim M, Seberg AP, Lander ES, Chadwick BP, Aiden EL (2016) Deletion of DXZ4 on the human inactive X chromosome alters higher-order genome architecture. Proc Natl Acad Sci 113:E4504–E4512PubMedPubMedCentralCrossRefGoogle Scholar
  63. 63.
    Giorgetti L, Lajoie BR, Carter AC, Attia M, Zhan Y, Xu J, Chen CJ, Kaplan N, Chang HY, Heard E, Dekker J (2016) Structural organization of the inactive X chromosome in the mouse. Nature 535:575–579PubMedPubMedCentralCrossRefGoogle Scholar
  64. 64.
    Sado T, Sakaguchi T (2013) Species-specific differences in X chromosome inactivation in mammals. Reproduction 146:R131–R139PubMedCrossRefGoogle Scholar
  65. 65.
    Okamoto I, Patrat C, Thépot D, Peynot N, Fauque P, Daniel N, Diabangouaya P, Wolf J-P, Renard J-P, Duranthon V, Heard E (2011) Eutherian mammals use diverse strategies to initiate X-chromosome inactivation during development. Nature 472:370–374PubMedCrossRefGoogle Scholar
  66. 66.
    Casas-Delucchi CS, Brero A, Rahn H-P, Solovei I, Wutz A, Cremer T, Leonhardt H, Cardoso MC (2011) Histone acetylation controls the inactive X chromosome replication dynamics. Nat Commun 2:1–11CrossRefGoogle Scholar
  67. 67.
    Sato Y, Kujirai T, Arai R, Asakawa H, Ohtsuki C, Horikoshi N, Yamagata K, Ueda J, Nagase T, Haraguchi T, Hiraoka Y, Kimura A, Kurumizaka H, Kimura H (2016) A genetically encoded probe for live-cell imaging of H4K20 monomethylation. J Mol Biol 428:3885–3902PubMedCrossRefGoogle Scholar
  68. 68.
    Hiratani I, Gilbert DM (2010) Autosomal lyonization of replication domains during early mammalian development. Adv Exp Med Biol 695:41–58PubMedCrossRefGoogle Scholar
  69. 69.
    Hiratani I, Ryba T, Itoh M, Hiratani I, Ryba T, Itoh M, Rathjen J, Kulik M, Papp B, Fussner E, Bazett-jones DP, Plath K, Dalton S, Rathjen PD, Gilbert DM (2010) Genome-wide dynamics of replication timing revealed by in vitro models of mouse embryogenesis. Genome Res 20:155–169PubMedPubMedCentralCrossRefGoogle Scholar
  70. 70.
    Ryba T, Hiratani I, Sasaki T, Battaglia D, Kulik M, Zhang J, Dalton S, Gilbert DM (2011) Replication timing: a fingerprint for cell identity and pluripotency. PLoS Comput Biol 7:e1002225PubMedPubMedCentralCrossRefGoogle Scholar
  71. 71.
    Pope BD, Ryba T, Dileep V, Yue F, Wu W, Denas O, Vera DL, Wang Y, Hansen RS, Canfield TK, Thurman RE, Cheng Y, Gülsoy G, Dennis JH, Snyder MP, Stamatoyannopoulos JA, Taylor J, Hardison RC, Kahveci T, Ren B, Gilbert DM (2014) Topologically associating domains are stable units of replication-timing regulation. Nature 515:402–405PubMedPubMedCentralCrossRefGoogle Scholar
  72. 72.
    Rao SSP, Huntley MH, Durand NC, Stamenova EK, Bochkov ID, Robinson JT, Sanborn AL, Machol I, Omer AD, Lander ES, Aiden EL (2014) A 3D map of the human genome at kilobase resolution reveals principles of chromatin looping. Cell 159:1665–1680PubMedPubMedCentralCrossRefGoogle Scholar
  73. 73.
    Hanna J, Cheng AW, Saha K, Kim J, Lengner CJ, Soldner F, Cassady JP, Muffat J, Carey BW, Jaenisch R (2010) Human embryonic stem cells with biological and epigenetic characteristics similar to those of mouse ESCs. Proc Natl Acad Sci USA 107:9222–9227PubMedPubMedCentralCrossRefGoogle Scholar
  74. 74.
    Chan YS, Göke J, Ng JH, Lu X, Gonzales KAU, Tan CP, Tng WQ, Hong ZZ, Lim YS, Ng HH (2013) Induction of a human pluripotent state with distinct regulatory circuitry that resembles preimplantation epiblast. Cell Stem Cell 13:663–675PubMedCrossRefGoogle Scholar
  75. 75.
    Gafni O, Weinberger L, Mansour AA, Manor YS, Chomsky E, Ben-Yosef D, Kalma Y, Viukov S, Maza I, Zviran A, Rais Y, Shipony Z, Mukamel Z, Krupalnik V, Zerbib M, Geula S, Caspi I, Schneir D, Shwartz T, Gilad S, Amann-Zalcenstein D, Benjamin S, Amit I, Tanay A, Massarwa R, Novershtern N, Hanna JH (2013) Derivation of novel human ground state naive pluripotent stem cells. Nature 504:282–286PubMedCrossRefGoogle Scholar
  76. 76.
    Ware CB, Nelson AM, Mecham B, Hesson J, Zhou W, Jonlin EC, Jimenez-Caliani AJ, Deng X, Cavanaugh C, Cook S, Tesar PJ, Okada J, Margaretha L, Sperber H, Choi M, Blau CA, Treuting PM, Hawkins RD, Cirulli V, Ruohola-Baker H (2014) Derivation of naive human embryonic stem cells. Proc Natl Acad Sci USA 111:4484–4489PubMedPubMedCentralCrossRefGoogle Scholar
  77. 77.
    Takashima Y, Guo G, Loos R, Nichols J, Ficz G, Krueger F, Oxley D, Santos F, Clarke J, Mansfield W, Reik W, Bertone P, Smith A (2014) Resetting transcription factor control circuitry toward ground-state pluripotency in human. Cell 158:1254–1269PubMedPubMedCentralCrossRefGoogle Scholar
  78. 78.
    Theunissen TW, Powell BE, Wang H, Mitalipova M, Faddah DA, Reddy J, Fan ZP, Maetzel D, Ganz K, Shi L, Lungjangwa T, Imsoonthornruksa S, Stelzer Y, Rangarajan S, D’Alessio A, Zhang J, Gao Q, Dawlaty MM, Young RA, Gray NS, Jaenisch R (2014) Systematic identification of culture conditions for induction and maintenance of naive human pluripotency. Cell Stem Cell 15:471–487PubMedPubMedCentralCrossRefGoogle Scholar
  79. 79.
    Valamehr B, Robinson M, Abujarour R, Rezner B, Vranceanu F, Le T, Medcalf A, Lee TT, Fitch M, Robbins D, Flynn P (2014) Platform for induction and maintenance of transgene-free hiPSCs resembling ground state pluripotent stem cells. Stem Cell Rep 2:366–381CrossRefGoogle Scholar
  80. 80.
    Duggal G, Warrier S, Ghimire S, Broekaert D, Van Der Jeught M, Lierman S, Deroo T, Peelman L, Van Soom A, Cornelissen R, Menten B, Mestdagh P, Vandesompele J, Roost M, Slieker RC, Heijmans BT, Deforce D, De Sutter P, De Sousa Lopes SC, Heindryckx B (2015) Alternative routes to induce naïve pluripotency in human embryonic stem cells. Stem Cells 33:2686–2698PubMedCrossRefGoogle Scholar
  81. 81.
    Theunissen TW, Friedli M, He Y, Planet E, O’Neil RC, Markoulaki S, Pontis J, Wang H, Iouranova A, Imbeault M, Duc J, Cohen MA, Wert KJ, Castanon R, Zhang Z, Huang Y, Nery JR, Drotar J, Lungjangwa T, Trono D, Ecker JR, Jaenisch R (2016) Molecular criteria for defining the naive human pluripotent state. Cell Stem Cell 19:502–515PubMedPubMedCentralCrossRefGoogle Scholar
  82. 82.
    Zimmerlin L, Park TS, Zambidis ET (2017) Capturing human naïve pluripotency in the embryo and in the dish. Stem Cells Dev 26:1141–1161PubMedCrossRefGoogle Scholar
  83. 83.
    Mekhoubad S, Bock C, De Boer AS, Kiskinis E, Meissner A, Eggan K (2012) Erosion of dosage compensation impacts human iPSC disease modeling. Cell Stem Cell 10:595–609PubMedPubMedCentralCrossRefGoogle Scholar
  84. 84.
    Dandulakis MG, Meganathan K, Kroll KL, Bonni A, Constantino JN (2016) Complexities of X chromosome inactivation status in female human induced pluripotent stem cells a brief review and scientific update for autism research. J Neurodev Disord 8:1–7CrossRefGoogle Scholar
  85. 85.
    Vallot C, Huret C, Lesecque Y, Resch A, Oudrhiri N, Bennaceur-Griscelli A, Duret L, Rougeulle C, Bennaceur A, Duret L, Rougeulle C (2013) XACT, a long noncoding transcript coating the active X chromosome in human pluripotent cells. Nat Genet 45:239–241PubMedCrossRefGoogle Scholar
  86. 86.
    Vallot C, Ouimette J-F, Makhlouf M, Feraud O, Pontis J, Come J, Martinat C, Bennaceur-Griscelli A, Lalande M, Rougeulle C (2015) Erosion of X chromosome inactivation in human pluripotent cells initiates with XACT coating and depends on a specific heterochromatin landscape. Cell Stem Cell 16:533–546PubMedCrossRefGoogle Scholar
  87. 87.
    Sahakyan A, Kim R, Chronis C, Sabri S, Bonora G, Theunissen TW, Kuoy E, Langerman J, Clark AT, Jaenisch R, Plath K (2017) Human naive pluripotent stem cells model X chromosome dampening and X inactivation. Cell Stem Cell 20:87–101PubMedCrossRefGoogle Scholar
  88. 88.
    Hayashi K, Ohta H, Kurimoto K, Aramaki S, Saitou M (2011) Reconstitution of the mouse germ cell specification pathway in culture by pluripotent stem cells. Cell 146:519–532PubMedCrossRefGoogle Scholar
  89. 89.
    Buecker C, Srinivasan R, Wu Z, Calo E, Acampora D, Faial T, Simeone A, Tan M, Swigut T, Wysocka J (2014) Reorganization of enhancer patterns in transition from naive to primed pluripotency. Cell Stem Cell 14:838–853PubMedPubMedCentralCrossRefGoogle Scholar
  90. 90.
    Hayashi K, Ogushi S, Kurimoto K, Shimamoto S, Ohta H, Saitou M (2012) Offspring from oocytes derived from in vitro primordial germ cell-like cells in mice. Science 338:971–975PubMedCrossRefGoogle Scholar
  91. 91.
    Rathjen J, Lake JA, Bettess MD, Washington JM, Chapman G, Rathjen PD (1999) Formation of a primitive ectoderm like cell population, EPL cells, from ES cells in response to biologically derived factors. J Cell Sci 112:601–612PubMedGoogle Scholar
  92. 92.
    Toyooka Y, Shimosato D, Murakami K, Takahashi K, Niwa H (2008) Identification and characterization of subpopulations in undifferentiated ES cell culture. Development 135:909–918PubMedCrossRefGoogle Scholar
  93. 93.
    Smith A (2017) Formative pluripotency: the executive phase in a developmental continuum. Development 144:365–373PubMedPubMedCentralCrossRefGoogle Scholar
  94. 94.
    Kalkan T, Smith A (2014) Mapping the route from naive pluripotency to lineage specification. Philos Trans R Soc Lond B Biol Sci 369:20130540PubMedPubMedCentralCrossRefGoogle Scholar
  95. 95.
    Kalkan T, Olova N, Roode M, Mulas C, Lee HJ, Nett I, Reik W, Bertone P, Smith A (2016) Tracking the embryonic stem cell transition from ground state pluripotency. Development 144:1221–1234CrossRefGoogle Scholar
  96. 96.
    Kurimoto K, Yabuta Y, Hayashi K, Ohta H, Kiyonari H, Mitani T, Moritoki Y, Kohri K, Kimura H, Yamamoto T, Katou Y, Shirahige K, Saitou M (2015) Quantitative dynamics of chromatin remodeling during germ cell specification from mouse embryonic stem cells. Cell Stem Cell 16:517–532PubMedCrossRefGoogle Scholar
  97. 97.
    Shirane K, Kurimoto K, Yabuta Y, Yamaji M, Satoh J, Ito S, Watanabe A, Hayashi K, Saitou M, Sasaki H (2016) Global landscape and regulatory principles of DNA methylation reprogramming for germ cell specification by mouse pluripotent stem cells. Dev Cell 39:87–103PubMedCrossRefGoogle Scholar
  98. 98.
    Wray J, Kalkan T, Gomez-Lopez S, Eckardt D, Cook A, Kemler R, Smith A (2011) Inhibition of glycogen synthase kinase-3 alleviates Tcf3 repression of the pluripotency network and increases embryonic stem cell resistance to differentiation. Nat Cell Biol 13:838–845PubMedPubMedCentralCrossRefGoogle Scholar
  99. 99.
    Leeb M, Dietmann S, Paramor M, Niwa H, Smith A (2014) Genetic exploration of the exit from self-renewal using haploid embryonic stem cells. Cell Stem Cell 14:385–393PubMedPubMedCentralCrossRefGoogle Scholar
  100. 100.
    Leeb M, Wutz A (2011) Derivation of haploid embryonic stem cells from mouse embryos. Nature 479:131–134PubMedPubMedCentralCrossRefGoogle Scholar
  101. 101.
    Yang H, Shi L, Wang BA, Liang D, Zhong C, Liu W, Nie Y, Liu J, Zhao J, Gao X, Li D, Xu GL, Li J (2012) Generation of genetically modified mice by oocyte injection of androgenetic haploid embryonic stem cells. Cell 149:605–617PubMedCrossRefGoogle Scholar
  102. 102.
    Takahashi S, Lee J, Kohda T, Matsuzawa A, Kawasumi M, Kanai-Azuma M, Kaneko-Ishino T, Ishino F (2014) Induction of the G2/M transition stabilizes haploid embryonic stem cells. Development 141:3842–3847PubMedCrossRefGoogle Scholar
  103. 103.
    Choi HW, Joo JY, Hong YJ, Kim JS, Song H, Lee JW, Wu G, Scholer HR, Do JT (2016) Distinct enhancer activity of Oct4 in naive and primed mouse pluripotency. Stem Cell Rep 7:911–926CrossRefGoogle Scholar
  104. 104.
    Kobayashi S, Hosoi Y, Shiura H, Yamagata K, Takahashi S, Fujihara Y, Kohda T, Okabe M, Ishino F (2016) Live imaging of X chromosome reactivation dynamics in early mouse development can discriminate naïve from primed pluripotent stem cells. Development 143:2958–2964PubMedCrossRefGoogle Scholar
  105. 105.
    Hadjantonakis A-K, Gertsenstein M, Ikawa M, Okabe M, Nagy A (1998) Non-invasive sexing of preimplantation stage mammalian embryos. Nat Genet 19:220–222PubMedCrossRefGoogle Scholar
  106. 106.
    TeSlaa T, Chaikovsky AC, Lipchina I, Escobar SL, Hochedlinger K, Huang J, Graeber TG, Braas D, Teitell MA (2016) α-Ketoglutarate accelerates the initial differentiation of primed human pluripotent stem cells. Cell Metab 24:485–493PubMedPubMedCentralCrossRefGoogle Scholar
  107. 107.
    Mathieu J, Ruohola-Baker H (2017) Metabolic remodeling during the loss and acquisition of pluripotency. Development 144:541–551PubMedCrossRefGoogle Scholar
  108. 108.
    Zhou W, Choi M, Margineantu D, Margaretha L, Hesson J, Cavanaugh C, Blau CA, Horwitz MS, Hockenbery D, Ware C, Ruohola-Baker H (2012) HIF1α induced switch from bivalent to exclusively glycolytic metabolism during ESC-to-EpiSC/hESC transition. EMBO J 31:2103–2116PubMedPubMedCentralCrossRefGoogle Scholar
  109. 109.
    Sperber H, Mathieu J, Wang Y, Ferreccio A, Hesson J, Xu Z, Fischer KA, Devi A, Detraux D, Gu H, Battle SL, Showalter M, Valensisi C, Bielas JH, Ericson NG, Margaretha L, Robitaille AM, Margineantu D, Fiehn O, Hockenbery D, Blau CA, Raftery D, Margolin AA, Hawkins RD, Moon RT, Ware CB, Ruohola-Baker H (2015) The metabolome regulates the epigenetic landscape during naive-to-primed human embryonic stem cell transition. Nat Cell Biol 17:1523–1535PubMedPubMedCentralCrossRefGoogle Scholar
  110. 110.
    Carey BW, Finley LWS, Cross JR, Allis CD, Thompson CB (2015) Intracellular α-ketoglutarate maintains the pluripotency of embryonic stem cells. Nature 518:413–416PubMedCrossRefGoogle Scholar
  111. 111.
    Shiraki N, Shiraki Y, Tsuyama T, Obata F, Miura M, Nagae G, Aburatani H, Kume K, Endo F, Kume S (2014) Methionine metabolism regulates maintenance and differentiation of human pluripotent stem cells. Cell Metab 19:780–794PubMedCrossRefGoogle Scholar
  112. 112.
    Hikabe O, Hamazaki N, Nagamatsu G, Obata Y, Hirao Y, Hamada N, Shimamoto S, Imamura T, Nakashima K, Saitou M, Hayashi K (2016) Reconstitution in vitro of the entire cycle of the mouse female germ line. Nature 539:299–303PubMedCrossRefGoogle Scholar
  113. 113.
    Harrison SE, Sozen B, Christodoulou N, Kyprianou C, Zernicka-goetz M (2017) Assembly of embryonic and extra-embryonic stem cells to mimic embryogenesis in vitro. Science 356:eaal1810PubMedCrossRefGoogle Scholar
  114. 114.
    Rotem A, Ram O, Shoresh N, Sperling RA, Goren A, Weitz DA, Bernstein BE (2015) Single-cell ChIP-seq reveals cell subpopulations defined by chromatin state. Nat Biotechnol 33:1165–1172PubMedPubMedCentralCrossRefGoogle Scholar
  115. 115.
    Farlik M, Sheffield NC, Nuzzo A, Datlinger P, Schönegger A, Klughammer J, Bock C (2015) Single-cell DNA methylome sequencing and bioinformatic inference of epigenomic cell-state dynamics. Cell Rep 10:1386–1397PubMedPubMedCentralCrossRefGoogle Scholar
  116. 116.
    Gravina S, Dong X, Yu B, Vijg J, Gravina S, Ganapathi S, Vijg J, Feinberg J, Toner C, Greenland P, Gidding S, Tracy R, Jaenisch R, Bird A, Deaton A, Bird A, Li W, Calder R, Mar J, Vijg J, Dey S, Kester L, Spanjaard B, Bienko M, Oudenaarden A, Faggioli F, Sacco M, Susani L, Montagna C, Vezzoni P, Krueger F, Andrews S (2016) Single-cell genome-wide bisulfite sequencing uncovers extensive heterogeneity in the mouse liver methylome. Genome Biol 17:1–8CrossRefGoogle Scholar
  117. 117.
    Buenrostro JD, Wu B, Litzenburger UM, Ruff D, Gonzales ML, Snyder MP, Chang HY, Greenleaf WJ (2015) Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523:486–490PubMedPubMedCentralCrossRefGoogle Scholar
  118. 118.
    Nagano T, Lubling Y, Stevens TJ, Schoenfelder S, Yaffe E, Dean W, Laue ED, Tanay A, Fraser P (2013) Single-cell Hi-C reveals cell-to-cell variability in chromosome structure. Nature 502:59–64PubMedCrossRefGoogle Scholar
  119. 119.
    Stevens TJ, Lando D, Basu S, Liam P, Cao Y, Lee SF, Leeb M, Wohlfahrt KJ, Boucher W, Shaughnessy-kirwan AO, Cramard J, Faure AJ, Ralser M, Blanco E, Morey L, Sansó M, Palayret MGS, Lehner B, Di Croce L, Wutz A, Hendrich B (2017) 3D structures of individual mammalian genomes studied by single-cell Hi-C. Nature 544:59–64PubMedPubMedCentralCrossRefGoogle Scholar
  120. 120.
    Flyamer IM, Gassler J, Imakaev M, Ulyanov SV, Abdennur N, Razin SV, Mirny L, Tachibana-Konwalski K (2017) Single-cell Hi-C reveals unique chromatin reorganization at oocyte-to-zygote transition. Nature 544:110–114PubMedPubMedCentralCrossRefGoogle Scholar
  121. 121.
    Kind J, Pagie L, De Vries SS, Nahidiazar L, Dey SS, Bienko M, Zhan Y, Lajoie B, De Graaf CA, Amendola M, Fudenberg G, Imakaev M, Mirny LA, Jalink K, Dekker J, Van Oudenaarden A, Van Steensel B (2015) Genome-wide maps of nuclear lamina interactions in single human cells. Cell 163:134–147PubMedPubMedCentralCrossRefGoogle Scholar
  122. 122.
    Clark SJ, Lee HJ, Smallwood SA, Kelsey G, Reik W (2016) Single-cell epigenomics: powerful new methods for understanding gene regulation and cell identity. Genome Biol 17:72PubMedPubMedCentralCrossRefGoogle Scholar
  123. 123.
    Wu J, Huang B, Chen H, Yin Q, Liu Y, Xiang Y, Zhang B, Liu B, Wang Q, Xia W, Li W, Li Y, Peng X, Zheng H, Ming J, Zhang W, Zhang J, Tian G, Xu F, Chang Z, Na J, Yang X, Xie W (2016) The landscape of accessible chromatin in mammalian preimplantation embryos. Nature 534:652–657PubMedCrossRefGoogle Scholar
  124. 124.
    Guo H, Zhu P, Wu X, Li X, Wen L, Tang F (2013) Single-cell methylome landscapes of mouse embryonic stem cells and early embryos analyzed using reduced representation bisulfite sequencing. Genome Res 23:2126–2135PubMedPubMedCentralCrossRefGoogle Scholar
  125. 125.
    Guo H, Zhu P, Yan L, Li R, Hu B, Lian Y, Yan J, Ren X, Lin S, Li J, Jin X, Shi X, Liu P, Wang X, Wang W, Wei Y, Li X, Guo F, Wu X, Fan X, Yong J, Wen L, Xie SX, Tang F, Qiao J (2014) The DNA methylation landscape of human early embryos. Nature 511:606–610PubMedCrossRefGoogle Scholar
  126. 126.
    Petropoulos S, Edsgärd D, Reinius B, Deng Q, Panula SP, Codeluppi S, Reyes AP, Linnarsson S, Sandberg R, Lanner F (2016) Single-cell RNA-Seq reveals lineage and X chromosome dynamics in human preimplantation embryos. Cell 165:1012–1026PubMedPubMedCentralCrossRefGoogle Scholar
  127. 127.
    Deng Q, Ramskold D, Reinius B, Sandberg R (2014) Single-cell RNA-seq reveals dynamic, random monoallelic gene expression in mammalian cells. Science 343:193–196PubMedCrossRefGoogle Scholar
  128. 128.
    Blakeley P, Fogarty NME, Del Valle I, Wamaitha SE, Hu TX, Elder K, Snell P, Christie L, Robson P, Niakan KK (2015) Defining the three cell lineages of the human blastocyst by single-cell RNA-seq. Development 142:3151–3165PubMedPubMedCentralCrossRefGoogle Scholar
  129. 129.
    Nakamura T, Okamoto I, Sasaki K, Yabuta Y, Iwatani C, Tsuchiya H, Seita Y, Nakamura S, Yamamoto T, Saitou M (2016) A developmental coordinate of pluripotency among mice, monkeys and humans. Nature 537:57–62PubMedCrossRefGoogle Scholar
  130. 130.
    Chen Y, Niu Y, Li Y, Ai Z, Kang Y, Shi H, Xiang Z, Yang Z, Tan T, Si W, Li W, Xia X, Zhou Q, Ji W, Li T (2015) Generation of cynomolgus monkey chimeric fetuses using embryonic stem cells. Cell Stem Cell 17:116–124PubMedCrossRefGoogle Scholar

Copyright information

© The Author(s) 2017

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.

Authors and Affiliations

  1. 1.Laboratory for Developmental EpigeneticsRIKEN Center for Developmental BiologyKobeJapan
  2. 2.Molecular Profiling Research Center for Drug DiscoveryNational Institute of Advanced Industrial Science and TechnologyTokyoJapan
  3. 3.Department of Epigenetics, Medical Research InstituteTokyo Medical and Dental UniversityTokyoJapan

Personalised recommendations